We report the initial characterization of adeno-associated virus type 5 (AAV5) RNAs generated following viral infection and the construction of a replicating infectious clone of AAV5. While the basic transcription profile of AAV5 was similar to that of AAV2, there were also significant differences. Mapping of the AAV5 transcripts demonstrated an efficient transcription initiation site within the AAV5 inverted terminal repeat (ITR), and mapping of the AAV5 intron revealed that it is considerably smaller than that of AAV2. Furthermore, in contrast to the case for AAV2, neither the Rep protein nor additional adenovirus gene products were required to achieve efficient promoter activity and pre-mRNA splicing following transfection of an AAV5 rep/cap plasmid clone lacking the ITRs into 293 cells. Perhaps most surprisingly, RNAs generated from both the AAV5 P7 and P19 promoters were efficiently polyadenylated at a site lying within the intronic region in the center of the genome. Because P7-and P19-generated transcripts are polyadenylated at this site and not spliced, Rep78 and Rep52 were the only Rep proteins detected during AAV5 infection.The human adeno-associated viruses (AAV) are small, nonenveloped, single-stranded DNA viruses that replicate in mammalian cells best in the presence of larger helper DNA viruses, e.g., adenovirus or herpesvirus (36). Six different serotypes of AAV (AAV type 1 [AAV1] to AAV6) have been characterized. AAV2, the prototypical strain, as well as AAV3 and AAV5 have been isolated directly from human clinical specimens (5, 12, 26). AAV1 and AAV4 have been suggested to be originally of simian origin (5, 26). The more distantly related AAV5 was isolated was from a penile flat condylomatous lesion (2), and epidemiologically, AAV5 transmission appears to follow acquisition of herpesviruses rather than adenovirus (12).At least portions of the genomes of all six serotypes of AAV have been cloned and sequenced (9,10,22,31,37,43). The inverted terminal repeats (ITRs) of AAV1, -2, -3, -4, and -6 are Ͼ95% identical, and the rep genes of these different isolates are approximately 85% identical. In contrast, the rep gene and ITR of AAV5 are only 60% similar to those of other serotypes (9).The Rep proteins of AAV1, -2, -3, -4, and -6 can each support the production of recombinant AAV2 vectors (9, 30), suggesting that they share significant functional homology for replication. In contrast, AAV5 is unable to support replication of AAV2-based vectors (9, 30), most likely because AAV5 Rep processes a novel terminal resolution site (TRS) present only on AAV5 ITR (8).AAV2 is the best characterized of the AAV serotypes. The genome contains three promoters, P5, P19, and P40. The large Rep proteins (Rep78 and Rep68) and the small Rep proteins (Rep52 and Rep40), encoded from a large open reading frame in the left half of the genome, are generated from RNAs which derive from P5 and P19, respectively, and which polyadenylate near the right-hand ITR. Rep78 and -52 are generated from unspliced RNAs, and Rep68 and -40...